The completion of Human Genome project spurred the rapid growth of a new interdisciplinary field of proteomics which includes: identification and characterization of complete sets of proteins encoded by the genome, the synthesis of proteins, post-translational modifications, as well as detailed mapping of protein interaction at the cellular regulation level.
While 2-dimensional gel electrophoresis in combination with mass spectrometry still remains the dominant technology in proteomics study, the successful implantation and application of DNA microarray technology to gene profiling and gene discovery have prompted scientists to develop protein microarray technology and apply microchip based protein assays to the field of proteomics. For example, in WO 00/04382 and WO 00/04389, a method of fabricating protein microarrays is disclosed. A key element in the disclosure is a substrate consisting of a solid support coated with a monolayer of thin organic film on which protein or a protein capture agent can be immobilized.
Nitrocellulose membrane was widely used as a protein blotting substrate in Western blotting and enzyme linked immunosorbent assay (ELISA). In WO 01/40312 and WO 01/40803, antibodies are spotted onto a nitrocellulose membrane using a gridding robot device. Such spotted antibody microarrays on a nitrocellulose membrane substrate have been shown to be useful in analyzing protein mixture in a large parallel manner.
In WO 98/29736, L. G. Mendoza et al. describe an antibody microarray with antibody immobilized onto a N-hydroxysuccinimidyl ester modified glass substrate. In U.S. Pat. No. 5,981,734 and WO 95/04594, a polyacrylamide based hydrogel substrate technology is described for the fabrication of DNA microarrays. More recently, in Anal. Biochem. (2000) 278, 123–131, the same hydrogel technology was further demonstrated as useful as a substrate for the immobilization of proteins in making protein microarrays.
In the above cited examples, the common feature among these different approaches is the requirement of a solid support that allows covalent or non-covalent attachment of a protein or a protein capture agent on the surface of said support. In DNA microarray technology, a variety of surfaces have been prepared for the deposition of pre-synthesized oligos and PCR prepared cDNA probes. For example, in EP 1 106 603 A2 a method of preparing vinylsulfonyl reactive groups on the surface to manufacture DNA chip is disclosed. In the preferred embodiments in EP 1 106 603 A2, silane coupling agents such as γ-aminopropyltrimethoxysilane are affixed to a glass substrate, referred to as a solid carrier, via covalent bonds to the silyloxy functional group. The pendant primary amino groups are then reacted with vinylsulfonyl compounds such as bis(vinylsulfonyl)methane (BVSM) to provide a reactive surface for attachment of DNA molecules. However, a limitation of this approach is that the reactions of primary amines with bis(vinylsulfones) like BVSM can lead to cyclizations via intramolecular Michael additions (reviewed in Little, R. D.; Masjedizadeh, M. R.; Wallquist, O.; McLoughlin, J. I., Organic Reactions; New York; Wiley, 1995, pp. 315–552), which result in the loss of both the amino and the vinylsulfonyl group. An example of a cyclization reaction involving a primary amine and a BVSM derivative has also been reported (Buchi, J.; Fueg, H. R.; Aebi, A. Helv. Chim. Acta 1959, 42, 1368–1374).
Moreover, even though the invention in EP 1 106 603 A2 may be useful in preparing DNA chip, it is not suitable for protein microarray applications. Unlike DNA, proteins tend to bind to surfaces in a non-specific manner and, in doing so, lose their biological activity. Thus, the attributes for a protein microarray substrate are different from those for a DNA microarray substrate in that the protein microarray substrate must not only provide surface functionality that are capable of interacting with protein capture agents, but must also resist non-specific protein binding to areas where no protein capture agents have been deposited.
Bovine serum albumin (BSA) has been demonstrated to be a useful reagent in blocking proteins from non-specific surface binding. Polyethylene glycol and phospholipids have also been used to passivate surfaces and provide a surface resistant to non-specific binding. However, all of these methods suffer disadvantages either because surface preparation takes a long time or because the method of surface modification is complex and difficult, making the method less than an ideal choice for large scale industrial manufacture.
U.S. Ser. No. 10/020,747 describes a low cost method of making protein microarray substrate using gelatin coating to create a reactive surface for immobilization of protein capture agents. While the gelatin modified surface effectively eliminates non-specific protein binding, the number of reactive sites on the surface are limited by the intrinsic functional groups in gelatin and the type of chemical agents (A-L-B) employed. Since the number of reactive sites on the surface directly determines the ultimate signal detection limit, it is desirable to create a surface with higher number of reactive sites that serves as a matrix on a solid support for the attachment of protein capture agents. The art needs a substrate with chemical functionality for the immobilization of protein capture agents, but such substrate must not bind proteins to areas on the gelatin surface that are without immobilized protein capture agents.
USSN 2003/0170474 A1 describes combinations of polymers, gelatin, and crosslinkers that are claimed to provide improved preparations of protein arrays. The polymers are referred to as scaffolds and are claimed to operate by effectively increasing the density of reactive functional crosslinking groups, such as activated olefins generated on reaction with a crosslinker molecule. These polymers are categorized into two general groups differing in their so-called crosslinking strategies. In one, polymers that contain functional groups that are reactive towards crosslinkers are claimed. Polymers containing groups including phosphines, thiols, and primary and secondary amines, are claimed. These polymers are proposed to increase the density of the desired attached proteins via conversion of these functional groups into groups that are capable of immobilizing proteins. The conversion of the functional groups is claimed to take place via reaction with a bifunctional crosslinking agent. In practice, the combinations of polymers based on the primary amine N-(3-aminopropyl)methacrylamide monomer and poly(ethylene)imine are cited. In addition, polymers containing the nitrogen acid, imidazole, e.g., poly(N-vinylimidazole) are claimed. A second strategy claimed in USSN 2003/0170474 A1 employs polymers which contain functions that are capable of immobilizing proteins, without the addition of a bifunctional agent.
It has since been found that some functional groups, such as nitrogen-containing compounds like amines, exhibit large variations in their reactivities towards certain classes of bifunctional crosslinkers, such as activated olefins like bis(vinylsulfonyl)methane (BVSM). Amines that exhibit low reactivity towards bifunctional crosslinkers are undesirable because they increase the time required to prepare the surface. Moreover, as cited above, certain types of functional groups, such as primary amines, react with crosslinkers like bis(vinylsulfonyl)methane to provide undesirable side products. These side reactions effectively remove both the functional group and the crosslinking agent, decreasing the efficiency of the process and the density of the functional groups. The present invention is designed to provide improvements over the existing art through the use of elements that contain piperazine functional groups that demonstrate improved reactivity towards bifunctional crosslinkers like bis(vinylsulfonyl)methane and are not subject to undesired side reactions such as intramolecular cyclization reactions.